Chloride is a known regulator of ion transporters, yet chloride sensors that control transport have not been identified in mammals. We discovered that the WNK [with no lysine (K)] kinase, WNK1, known to be salt sensitive, is directly regulated by chloride, and found the chloride binding site in structural studies. Here we will determine whether one or all WNKs (isoforms 1, 2, 3, and 4) are regulated by chloride, and over what concentration ranges, using assays, thermal stability measurements and isothermal tritration calorimetry. We will determine how chloride affects the structure of the kinase domain of WNK1 through structural comparisons of chloride-bound and active WNK1, with existing crystals. WNKs are regulated by phosphorylation and autoinhibition. How the chloride inhibition relates to these additional mechanisms will be assessed by assays and crystallography. Mutants affecting chloride binding will be designed and evaluated. The WNK-based chloride sensing mechanism will be tested in cells.